Methods for maintaining hepatocytes in culture and for differentiating embryonic stem cells along a hepatocyte lineage
First Claim
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1. A cell differentiated from an embryonic stem (ES) cell along a hepatocyte lineage by culturing said ES cell in a medium with growth factors consisting essentially of insulin and dexamethasone.
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Abstract
The invention provides methods and media for culturing embryonic stem (ES) cells, such as human ES cells, and directing them along the hepatic lineage. It further provides methods for maintaining hepatocytes in culture for extended periods. The invention further provides cells cultured by the methods of the invention. Additionally, the invention provides methods of transducing cells with marker proteins that will be expressed only in hepatocyte-like cells and selecting for cells expressing the marker protein.
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111 Claims
- 1. A cell differentiated from an embryonic stem (ES) cell along a hepatocyte lineage by culturing said ES cell in a medium with growth factors consisting essentially of insulin and dexamethasone.
- 18. An isolated hepatocyte maintained in culture by culturing said hepatocyte in a medium with growth factors consisting essentially of insulin and dexamethasone.
- 34. A method of differentiating embryonic stem (ES) cells along a hepatocyte lineage, said method comprising culturing said ES cell in a medium with growth factors consisting essentially of insulin and dexamethasone.
- 51. A method of maintaining a hepatocyte in culture for an extended period, said method comprising culturing said hepatocyte cell in a medium in which growth factors consist essentially of insulin and dexamethasone.
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67. A method of screening a compound for its effects on a hepatocyte or on a hepatocyte activity, said method comprising
(a) contacting the compound to a cell selected from the group consisting of (i) an embryonic stem (ES) cell differentiated along a hepatocyte lineage by culturing said ES cell with a culture medium containing growth factors, wherein said growth factors consist essentially of insulin and dexamethasone, and (ii) an isolated hepatocyte maintained in culture in a culture medium containing growth factors, wherein said growth factors consist essentially of insulin and dexamethasone; -
(b) determining any change to the cells of step (a) contacted with said compound or in an activity of said cells of step (a) contacted with said compound; and
(c) correlating the change of step (b) with the effect of the compound on a cell of step (a) or on an activity of said cell. - View Dependent Claims (68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79)
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- 80. A cell culture for producing one or more liver proteins, said cell culture selected from the group consisting of (i) an embryonic stem (ES) cell differentiated along a hepatocyte lineage by culturing said ES cell with a culture medium containing growth factors, wherein said growth factors consist essentially of insulin and dexamethasone, (ii) an isolated hepatocyte maintained in culture in a culture medium containing growth factors, wherein said growth factors consist essentially of insulin and dexamethasone, and (iii) a combination of cells of (a) and (b).
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96. A method of producing a liver protein, comprising
(a) providing a cell culture selected from the group consisting of (i) a culture of embryonic stem (ES) cells differentiated along a hepatocyte lineage by culturing said ES cells with a culture medium containing growth factors, wherein said growth factors consist essentially of insulin and dexamethasone, (ii) isolated hepatocytes maintained in culture in a culture medium containing growth factors, wherein said growth factors consist essentially of insulin and dexamethasone, and (iii) a combination of cells of (i) and (ii); - and
(b) isolating said liver protein from said culture. - View Dependent Claims (97, 98, 99, 100, 101, 102, 103, 104, 106, 107, 108)
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105. A method of claini 96, wherein the cell of step (a) is cultured on an extracellular matrix of collagen type 1.
- 109. A method for identifying cells expressing a hepatocyte-like phenotype, said method comprising transducing a population of cells with a lentiviral vector comprising a gene encoding a marker protein, wherein said gene is operably linked to a promoter for proteins exclusively or preferentially expressed in hepatocytes, and identifying cells expressing the marker protein.
Specification